An enzymatic process for the production of a diglyceride oil

Abstract

The application discloses an enzymatic production process of diglyceride, and comprises the following steps: using low-cost lipase as a catalyst, preparing lipase A through immobilization of the enzyme, preparing lipase B through an ester exchange reaction, and then using the lipase B as a catalyst to catalyze esterification of free fatty acid and glycerol to produce diglyceride; the method can realize efficient enzymatic production of diglyceride through a series of methods by using low-cost lipase, and the production cost is reduced. The application provides an economical and efficient method for producing diglyceride by using the enzyme method, greatly reduces the use cost of lipase in the enzymatic esterification reaction, and has certain industrialization application prospect.

Description

Technical Field

[0001] This invention belongs to the field of food processing technology, specifically relating to an enzymatic production technology and process for functional oils. Background Technology

[0002] Diacylglycerol (DAG) is a molecule formed by the esterification of two of the three hydroxyl groups on the glycerol backbone with fatty acids. In natural animal and plant oils, diglycerides are present in trace amounts, generally not exceeding 1%. Recent animal and human studies have shown that oils rich in diglycerides have a range of benefits, including weight loss, lowering blood lipids, preventing atherosclerosis, and reducing uric acid levels, making them a highly valuable functional oil for development.

[0003] Currently, enzymatic methods for preparing diglycerides include enzymatic hydrolysis, enzymatic glycerol hydrolysis, and enzymatic esterification.Enzymatic hydrolysis produces a large amount of free fatty acids as a byproduct while generating diglycerides. Therefore, compared with enzymatic glycerol hydrolysis and esterification, the yield of diglyceride oil produced by enzymatic hydrolysis is low. Enzymatic esterification, on the other hand, utilizes the esterification reaction of free fatty acids, a byproduct of oil processing, with glycerol to produce diglyceride oil, thus making efficient use of byproduct resources. Generally, the lipases used in enzymatic esterification are usually derived from Candida antarctica (Lipozyme 435 and Novozym 435), Rhizomucor miehei (Lipozyme RM IM and Lipozyme RM), and Rhizopus oryzae. Lipases derived from Thermomyces lanuginosus, such as Lipozyme TL 100L and Lipozyme TLIM (an immobilized version of Lipozyme TL 100L), generally have no activity in the esterification reaction system of glycerol and fatty acids (Guo and Sun, Solvent-free enzymatic synthesis of 1,3-diconjugated linoleoylglycerol optimized by response surface methodology, Biotechnology Progress, 2004, 20, 619−622; Li Yue et al., Research progress in enzymatic preparation of diglycerides, China Oils and Fats, 2022, 47, 77-84; Guo and Sun, Solvent-free production of 1,3-diglyceride of CLA: Strategy consideration and protocol design. Food Chemistry, 2007, 100 1076-1084; Lu Shan, Preparation of high-purity 1,3-dioleoylglycerol, Master's thesis of Jiangnan University, 2013), but lipases derived from Thermomyces lanuginosus are generally very inexpensive. For example, Lipozyme TL IM is about 10% of the price of Lipozyme RM, and Lipozyme 435 is about 5% of the price of Lipozyme 435; the price of Lipozyme TL 100L is about the same as that of Lipozyme 435. With RM accounting for 5% and Lipozyme 435 for 2%, the urgent problem to be solved is how to prepare diglyceride oil by enzymatic esterification using an inexpensive lipase (such as lipases derived from Thermomyces lanuginosus and Aspergillus niger).

[0004] We still need to find an economical and efficient enzymatic production process for diglycerides, utilizing commercially available and inexpensive lipases to catalyze the esterification reaction of glycerol and free fatty acids, and then separate functional oils rich in diglycerides. Summary of the Invention

[0005] The purpose of this section is to outline some aspects of embodiments of the present invention and to briefly describe some preferred embodiments. Simplifications or omissions may be made in this section, as well as in the abstract and title of this application, to avoid obscuring the purpose of these documents; however, such simplifications or omissions should not be construed as limiting the scope of the invention.

[0006] In view of the problems existing in the above and / or prior art, the present invention is proposed.

[0007] One objective of this invention is to provide an economical and efficient enzymatic production process for diglyceride oil. The process involves preparing diglyceride oil through enzymatic esterification, using relatively inexpensive commercial enzymes as catalysts to catalyze the esterification reaction between free fatty acids and glycerol, thereby producing oils rich in diglycerides. Before the enzymatic esterification reaction, the lipase is immobilized and subjected to random transesterification to improve its catalytic efficiency in the esterification reaction system.

[0008] To solve the above-mentioned technical problems, the present invention provides the following technical solution: an enzymatic production process for diglyceride oil, the production process comprising the following steps: Step (1) Immobilization of lipase: Immobilize lipase on a nonpolar carrier to prepare immobilized lipase A; The lipase is derived from one or more of Thermomyces lanuginosus or Aspergillus niger; the nonpolar carrier is a BS-type material, D101, or X-5. Step (2) Transesterification reaction: The immobilized lipase A obtained in step (1) is mixed with animal and vegetable oils and transesterified at 35-65°C. This step is repeated until the acid value of the transesterified animal and vegetable oils drops to 1.5 mgKOH / g or below, and the immobilized lipase B is separated from the animal and vegetable oils. The animal and vegetable oils do not include fish oil, algal oil, and oils with high saturation rich in DHA and EPA. The acid value of the animal and vegetable oils is less than 0.2 mgKOH / g. Step (3) Esterification reaction: Add the immobilized lipase B and polar adsorbent obtained in step (2) to the free fatty acids and glycerol, and carry out the esterification reaction at 35-65℃. After the crude product is deacidified by distillation, the diglyceride oil product is obtained.

[0009] As a preferred embodiment of the enzymatic production process of diglyceride oil of the present invention, maltodextrin is also added during the lipase immobilization process in step (1).

[0010] As a preferred embodiment of the enzymatic production process of diglyceride oil of the present invention, the amount of maltodextrin added is 8%-15% (based on the total substrate mass).

[0011] As a preferred embodiment of the enzymatic production process of diglyceride oil of the present invention, step (1) specifically involves diluting the concentration of lipase solution to 15 mg / mL with a buffer solution on a nonpolar carrier, setting the pH of the enzyme solution to 5.5, the temperature to 25°C, and adding 10% maltodextrin. After adsorption for 6 h, the unadsorbed free enzyme is removed with a buffer solution, and the immobilized enzyme is obtained after vacuum drying.

[0012] As a preferred embodiment of the enzymatic production process of diglyceride oil of the present invention, in step (2), the transesterification reaction temperature is 40-55℃ and the reaction time is 2-6 h.

[0013] As a preferred embodiment of the enzymatic production process of diglyceride oil of the present invention, in step (3), the free fatty acid is a C8-C18 carbon chain fatty acid.

[0014] As a preferred embodiment of the enzymatic production process of diglyceride oil of the present invention, in step (3), the molar ratio of free fatty acids to glycerol is (0.8-2):1.

[0015] As a preferred embodiment of the enzymatic production process of diglyceride oil of the present invention, in step (3), the esterification reaction is carried out under vacuum conditions and the pressure of the reaction system is less than 100 Pa.

[0016] As a preferred embodiment of the enzymatic production process of diglyceride oil of the present invention, the reaction temperature in step (3) is 45-65℃.

[0017] As a preferred embodiment of the enzymatic production process of diglyceride oil of the present invention, wherein the polar adsorbent in step (3) includes silica gel, Amberlite FPA54 and S-8.

[0018] Another object of the present invention is to provide the application of the enzymatic production process of diglyceride oil as described in any of the above claims in the food and pharmaceutical fields.

[0019] Compared with the prior art, the present invention has the following beneficial effects: This invention improves the catalytic activity of lipases derived from Thermomyces lanuginosus and Aspergillus niger in the esterification reaction system of free fatty acids and glycerol by immobilizing and transesterifying them, thus solving the problem of high enzyme cost in the esterification production of diglycerides and showing broad application prospects. Attached Figure Description

[0020] Figure 1 (a) is a liquid chromatogram of the DAG content in the esterification reaction product catalyzed by lipase TLL-BS55-B3 under the conditions of Example 2; (b) is a liquid chromatogram of the purified product obtained by molecular distillation of the esterification reaction product catalyzed by lipase TLL-BS55-B3 under the conditions of Example 2. Detailed Implementation

[0021] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to the examples in the specification.

[0022] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways different from those described herein, and those skilled in the art can make similar extensions without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below.

[0023] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The phrase "in one embodiment" appearing in different places in this specification does not necessarily refer to the same embodiment, nor is it a single or selective embodiment that is mutually exclusive with other embodiments.

[0024] Unless otherwise specified, all raw materials used in the examples are commercially available.

[0025] Lipase immobilization: 0.5 g of resin was used to dilute the enzyme solution to a concentration of 15 mg / mL with buffer solution. The enzyme solution was kept at pH 5.5 and temperature 25℃. 10% maltodextrin was added. After adsorption for 6 h, unadsorbed free enzyme was removed with buffer solution, and the immobilized enzyme was obtained by vacuum drying. Immobilized lipase A, abbreviated as TLL-BS55-A, was immobilized on BS-55 using Thermomyces lanuginosus Lipase (TLL) (or Aspergillusniger lipase, ANL) as the free enzyme. Other immobilized lipases were processed similarly.

[0026] The method for detecting diglyceride content is as follows: refer to the method in GB / T 26636.

[0027] Molecular distillation conditions: main evaporation temperature 190℃, condensation temperature 30℃, distillation pressure <1 Pa.

[0028] Example 1: Transesterification Effects of different animal and vegetable oils on transesterification: 100 g of animal or vegetable oil (acid value less than 0.2 mg / g) was weighed, and 10% (based on oil mass) of immobilized lipase TLL-BS55-A (lipase A) was added. Transesterification was carried out at 45℃ under vacuum. After 3 h of reaction, the immobilized lipase and the animal / vegetable oil were separated, and the acid value of the animal / vegetable oil was determined. If the acid value was above 1.5 mg / g, another 100 g of animal / vegetable oil was added to the separated TLL-BS55 enzyme for another 3 h of transesterification until the acid value of the animal / vegetable oil was less than 1.5 mg / g. The resulting immobilized lipase B (abbreviated as TLL-BS55-B) was used for subsequent esterification reactions. The number of transesterification cycles required for the acid value of the animal / vegetable oil to be less than 1.5 mg / g after transesterification with TLL-BS55-A in various animal and vegetable oils is shown in Table 1.

[0029] Table 1. Number of transesterification cycles required for the acid value of animal and vegetable oils to be less than 1.5 mg / g As shown in Table 1, for most oils with high unsaturation, the acid value of the oils obtained after three transesterifications is less than 1.5 mg / g. However, for oils with high saturation, such as palm oil and coconut oil, four transesterifications are required to obtain oils with an acid value less than 1.5 mg / g. Furthermore, although lipase A exhibits high esterification activity in the subsequent step (3) after transesterification, fish oil is an exception. The TLL-BS55-B obtained after transesterification in fish oil shows low activity in the esterification reaction. Therefore, soybean oil, rapeseed oil, and other oils with a certain degree of unsaturation (excluding oils rich in DHA) and immobilized lipase A were selected for transesterification.

[0030] Example 2: Transesterification and Esterification Reactions Effect of the number of transesterification reactions: 10% immobilized lipase TLL-BS55-A (or ANL-BS55-A) and soybean oil were mixed in a reactor and transesterified under 45°C and vacuum conditions. After 3 h of reaction, the soybean oil was separated, and fresh soybean oil was added to the reactor. This process was repeated three times. The enzyme TLL-BS55-B, which catalyzed 1-3 transesterifications of soybean oil, was collected and used to catalyze the esterification reaction of oleic acid and glycerol (Table 2). Soybean oil from 1-3 transesterifications was also collected, and the acid value of the soybean oil was determined (Table 2).

[0031] Esterification reaction: Oleic acid and glycerol were mixed in a molar ratio of 2:1, and silica gel was added to the mixture. The mass ratio of silica gel to glycerol was 1:1. The reaction temperature was 50℃, and the amount of immobilized lipase B added was 5% (based on the weight of oleic acid). The reaction was stopped after 8 h under vacuum conditions (pressure 50 Pa). The content of diglycerides in the crude product was analyzed and detected. The results are shown in Table 2.

[0032] Table 2. Acid value of soybean oil and catalyst performance of esterification reaction after different transesterification cycles. The results in the table above show that after three transesterifications in soybean oil matrix, the acid value of soybean oil becomes below 1.5 mg / g. Furthermore, this enzyme (TLL-BS55-B3) exhibits the best catalytic effect in esterification, with the crude product containing over 30% DAG. However, after one and two transesterifications, the acid value of soybean oil remains high. The lipases TLL-BS55-B1 (after one transesterification) and TLL-BS55-B2 (after two transesterifications) show weak catalytic activity in the esterification of oleic acid and glycerol, with the crude product containing less than 30% DAG. TLL-BS55 enzyme (lipase A), without transesterification, shows almost no catalytic activity when directly used to catalyze the esterification of glycerol and oleic acid. Similarly, ANL-BS55 exhibits very low catalytic activity when directly catalyzing esterification without transesterification.

[0033] Example 3: Transesterification and Esterification Reactions Influence of immobilization materials: Immobilized lipase A was prepared by immobilizing TLL enzyme on supports of different polarities. The nonpolar supports selected were BS-55, X-5, and D101; the medium polar supports were BS-30, ADS-17, and AB-8; and the polar supports were silica gel and NKA9. Immobilized lipase A was transesterified three times in soybean oil to obtain immobilized lipase B, which was used to catalyze the esterification reaction of oleic acid and glycerol. Other transesterification and esterification reaction conditions were the same as in Example 2.

[0034] Table 3. Effect of immobilized support on lipase B-catalyzed esterification reaction The results in the table above show that when lipase TLL from Thermomyces lanuginosus is immobilized on carriers of different polarities to obtain lipase A, and then undergoes three transesterification reactions to obtain lipase B immobilized on different carriers, the order of DAG content in the esterification reaction catalyzed by lipase B on different carriers is: lipase B immobilized on nonpolar material > lipase B immobilized on medium polar material > lipase B immobilized on polar material. In addition, when lipase ANL from Aspergillus niger is immobilized on a nonpolar carrier, its catalytic esterification reaction of oleic acid also has a high DAG content.

[0035] Example 4: Transesterification and Esterification Reactions Effect of transesterification temperature: ANL was immobilized on BS55 to obtain lipase ANL-BS55, and then soybean oil transesterification reaction was catalyzed at 35℃-65℃ (this process was repeated 3 times) to prepare lipase B under different temperature conditions. Other transesterification and esterification reaction conditions were the same as in Example 2. The DAG content in the crude product is shown in the table below.

[0036] Table 4. Effect of transesterification temperature on lipase B-catalyzed esterification reaction The results in the table above show that the optimal transesterification temperature range is 40-55℃.

[0037] Example 5: Transesterification and Esterification Reactions Effect of esterification temperature: ANL was immobilized on BS55 to obtain lipase ANL-BS55, and then the soybean oil transesterification reaction was catalyzed at 45℃ (this process was repeated 3 times) to prepare lipase ANL-BS55-B3. This enzyme was used to catalyze the esterification reaction at a temperature of 35℃-65℃. Other transesterification and esterification reaction conditions were the same as in Example 2. The DAG content in the crude product is shown in the table below.

[0038] Table 5. Effect of esterification temperature on lipase B-catalyzed esterification reaction The results in the table above show that the optimal esterification temperature is in the range of 45-65℃.

[0039] Example 6: Transesterification and Esterification Reactions Effect of substrate molar ratio in esterification reaction: The immobilized enzyme TLL-BS55-B3 obtained in Example 2 was used to catalyze the esterification reaction of oleic acid and glycerol. The molar ratio of oleic acid to glycerol was 0.5:1, 0.8:1, 1:1, 1.5:1, 2:1 and 2.5:1, respectively. Other conditions were the same as in Example 2. The DAG content in the crude product is shown in the table below.

[0040] Table 6. Effect of substrate molar ratio on lipase B-catalyzed esterification reaction The results in the table above show that the optimal molar ratio of oleic acid to glycerol is in the range of 0.8:1 to 2:1.

[0041] Example 7: Transesterification and Esterification Reactions Effect of fatty acid type on esterification reaction: The immobilized enzyme TLL-BS55-B3 obtained in Example 2 was used to catalyze the esterification reaction of glycerol and different fatty acids. The selected fatty acids were acetic acid, caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, oleic acid, linoleic acid, and DHA (docosahexaenoic acid). Other conditions were the same as in Example 2. The DAG content in the crude product is shown in the table below.

[0042] Table 7. Effect of fatty acid type on lipase B-catalyzed esterification reaction in esterification. The results in the table above show that the best lipases are those for C8-C18 fatty acids, with the enzyme showing the best specificity for lauric acid.

[0043] Example 8: Transesterification and Esterification Reactions Effects of Adsorbent Type and Polarity in Esterification: Immobilized lipase B, namely lipase TLL-D101-B3, was prepared according to the method in Example 3. This enzyme was used to catalyze the esterification reaction of oleic acid and glycerol. The effects of different adsorbents on the esterification reaction were investigated. The types of adsorbents investigated included: polar adsorbents (silica gel, Amberlite FPA54, S-8), moderately polar adsorbents (ADS-18, AB-8, BS-45), and non-polar adsorbents (D101, X-5, D1400, ADS-8). Other esterification conditions were the same as in Example 2. The DAG content in the crude product is shown in the table below.

[0044] Table 8. Effect of Adsorbent Type on Lipase B-Catalyzed Esterification Reaction in Esterification. The results in the table above show that adding a polar adsorbent to the esterification reaction yields the best results.

[0045] Comparative Example 1: Transesterification and Esterification The esterification reaction of oleic acid and glycerol was catalyzed using the commercially available immobilized lipase Lipozyme TL IM derived from Thermomyces lanuginosus as a catalyst. The esterification reaction conditions were the same as in Example 2. After the reaction, the DAG content in the crude product was measured to be 8.6%, while the DAG content in the esterification reaction catalyzed by the TLL-BS55-B3 lipase prepared by the method of the present invention was as high as 32.1%.

[0046] For all crude products with a DAG content of 30% or higher, after two-stage molecular distillation to remove free fatty acids and monoglycerides, the purified product had a DAG content greater than 90%. When the molar ratio of oleic acid to glycerol was 1:1 to 2:1, the purified crude product had a DAG content greater than 95%. Figure 1 (b)).

[0047] This invention employs an enzymatic esterification method to prepare diglyceride oil. Specifically, it uses a relatively inexpensive lipase as a biocatalyst to react free fatty acids and glycerol, producing an oil rich in diglycerides. Prior to the enzymatic esterification reaction, the lipase undergoes immobilization and random transesterification to improve its catalytic efficiency in the esterification reaction system, thereby reducing production costs and increasing the diglyceride content of the product. This invention provides an economical and efficient method for the enzymatic esterification of diglycerides, significantly reducing the cost of enzyme preparations for diglyceride production and demonstrating promising prospects for industrial application.

[0048] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. An enzymatic production process for diglyceride oil, characterized in that: The production process includes the following steps: Step (1) Immobilization of lipase: Immobilize lipase on a nonpolar carrier to prepare immobilized lipase A; The lipase is derived from one or more of Thermomyces lanuginosus or Aspergillus niger; the nonpolar carrier is a BS-type material, D101, or X-5. Step (2) Transesterification reaction: The immobilized lipase A obtained in step (1) is mixed with animal and vegetable oils and transesterified at 35-65°C. This step is repeated until the acid value of the transesterified animal and vegetable oils drops to 1.5 mgKOH / g or below, and the immobilized lipase B is separated from the animal and vegetable oils. The animal and vegetable oils do not include fish oil, algal oil, and oils with high saturation rich in DHA and EPA. The acid value of the animal and vegetable oils is less than 0.2 mgKOH / g. Step (3) Esterification reaction: Add the immobilized lipase B and polar adsorbent obtained in step (2) to the free fatty acids and glycerol, and carry out the esterification reaction at 35-65℃. After the crude product is deacidified by distillation, the diglyceride oil product is obtained.

2. The enzymatic production process for diglyceride oil as described in claim 1, characterized in that: In the lipase immobilization process described in step (1), maltodextrin is also added.

3. The enzymatic production process for diglyceride oil as described in claim 1 or 2, characterized in that: Step (1) specifically involves diluting the lipase solution concentration to 15 mg / mL with a buffer solution on a nonpolar carrier, setting the enzyme solution pH to 5.5 and the temperature to 25°C, and adding 10 wt% maltodextrin based on the immobilized carrier. After adsorption for 6 h, the unadsorbed free enzyme is removed with a buffer solution, and the immobilized enzyme is obtained after vacuum drying.

4. The enzymatic production process for diglyceride oil as described in claim 1, characterized in that: In step (2), the transesterification reaction temperature is 40–55 °C.

5. The enzymatic production process for diglyceride oil as described in claim 1, characterized in that: In step (3), the molar ratio of free fatty acids to glycerol is (0.8-2):

1.

6. The enzymatic production process for diglyceride oil as described in claim 1, characterized in that: In step (3), the free fatty acid is a C8-C18 carbon chain fatty acid.

7. The enzymatic production process for diglyceride oil as described in claim 1, characterized in that: In step (3), the esterification reaction is carried out under vacuum conditions and the pressure of the reaction system is less than 100 Pa.

8. The enzymatic production process for diglyceride oil as described in claim 1, characterized in that: The reaction temperature in step (3) is 45-65℃.

9. The enzymatic production process for diglyceride oil as described in claim 8, characterized in that: The polar adsorbents in step (3) include silica gel, Amberlite FPA54 and S-8.

10. The application of the enzymatic production process of diglyceride oil according to any one of claims 1 to 9 in the food and pharmaceutical fields.

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